Abstract In most cells the MHC I antigen presentation pathway exclusively displays peptides that are derived from a cell's own proteins. In contrast, dendritic cells (DCs) and macrophages (Ms) are capable of acquiring and then displaying peptides from external antigens through a process called cross presentation (XPT). XPT is the key mechanism that allows the immune system to recognize and then mobilize a CD8 T cell response to cancers, many viral infections and intracellular pathogens. Consequently, this pathway is important for immune surveillance and is an attractive target to enable vaccines to elicit CD8 T cell immunity, which is something current subunit vaccines fail to do. The overall goal of this grant is to elucidate key mechanisms that allow DCs to carry out this critical function. In the major XPT pathway, exogenous proteins are first internalized into phagosomes and then transferred into the cytosol, where they are cleaved into oligopeptides by proteasomes. Recent data suggests that the proteasome-generated peptides for XPT are subsequently imported back into phagosomes for binding to MHC I molecules (referred to here as ?phagosomal XPT?). However, why such peptides would not simply be delivered to MHC I molecules in the ER, as most cytosolic generated peptides are, is a mystery and one that our first aim seeks to solve. Our underlying hypothesis, supported by preliminary data, is that a subset of proteasomes physically associates with the cytosolic face of phagosomes and does so by via their PA28 capping complex binding to the cytosolic domains of the peptide-loading complex (TAP+Tapasin). This arrangement thereby links local peptide generation (by the phagosome-bound proteasomes) to local peptide transport (by phagosomal TAP). The importance of these hypotheses is that they have the potential to fill in key missing links in the phagosomal XPT pathway and to identify an important function for PA28 complexes, which up until now have thought to be relatively unimportant. Our second aim seeks to elucidate mechanisms that allow MHC I molecules to bind peptides in the ?unfriendly? environment of the phagosome and to address the question of why XPT of antigens is surprisingly much more efficient when the exogenous antigen is cell-associated, as compared to the same antigen in any other form. This aim will test the hypothesis that 2 microblobulin (2M) from ingested exogenous cells promotes the formation of peptide-MHC I complexes in phagosomes. In this mechanism, free 2M (from ingested cells) + free MHC I heavy chains delivered to and/or generated in phagosomes (from complexes denatured in the vacuole) + peptides (from PA28-proteasome-products that are imported into phagosomes by TAP) associate to form intraphagosomal MHC I-peptide complexes. The importance of this hypothesis is that it would provide insight into a biologically important process. Moreover, this mechanism may be able to be manipulated to enhance XPT for vaccines/immunotherapies.